Engineering Living Proteins

A general look at O-phosphoserine (Sep), the most abundant phosphoamino acid. Image courtesy of Sterling Professer Söll, DUS of the Department of Molecular Biochemistry and Biophysics.

When Watson and Crick revealed the structure of DNA in the 1950s, they sparked a revolutionary scientific expedition, one that has gone on for decades. Since then, scientists have sought to not only decipher the nature of the genetic code of life, but also manipulate the natural genetic code within organisms for synthesis biology. Yale University researchers, led by Professor of Molecular Biophysics and Biochemistry and Chemistry, Dieter Söll, along with Dr. Christopher Noren, head of the Division of Chemical Biology at New England Biolabs, and Assistant Professor of Physiology Jesse Rinehart, have taken this scientific expedition by storm by expanding the genetic code of Escherichia coli bacteria, gaining the ability to synthesize “special forms of proteins that can mimic proteins from natural or disease states.” By doing so, the research team now has tools available to study diseases in a completely new manner and lead the scientific revolution through the discovery of new drugs.

Simply put, the team has developed a method of influencing the functions of proteins as they are being created. The team induces phosphorylation, so that the protein’s function can be regulated through a method known as “phosphoregulation.” Essentially, phosphoregulation allows the switching of proteins “on and off ” so that certain disease states can be analyzed with more scrutiny than others. Because the rules for phosphoregulation are not directly encoded in the DNA, the Yale researchers engineered the E. coli’s genetic code to include phosphoserine, an ester of serine and phosphoric acid that will allow protein function to be regulated from the DNA itself.

Three steps to introduce the Phosphoserine into the genetic code of E. coli. Courtesy of Sterling Professor Söll, DUS Yale Department of Molecular Biochemistry and Biophysics

The goal of the project was to allow the co-translational insertion of O-phosphoserine (Sep), the most abundant phosphoamino acid related to protein phosphorylation. Sep is not encoded in the genetic code, but rather synthesized after translation in humans. For Sep insertion to occur successfully, the genetic code of the bacteria needed to be expanded, which requires the “freeing” of a codon from the normal genetic code. After a codon is “freed,” it is “reassigned” to permit the incorporation of a new amino acid. Typically, in order to allow for the “freeing of a codon,” UAG, a rare stop codon, is utilized; therefore, it seemed like the best option for the team. Next, as Rinehart, one of the lead researchers on the team, stated, “it was essential that the team repurpose the naturally-occurring enzyme SepRS (Sep tRNA Synthetase), [which is] responsible for delicately placing the phosphoserine onto tRNA.” This tRNA was then redesigned so that it was only recognized by SepRS, thus providing the aminoacylated-tRNA with phosphoserine and accomplishing the project’s primary goal. In normal translation, the process of delivering incorrectly aminoacylated-tRNAs to the ribosome where the mRNA interprets the order of amino acids is somewhat inefficient for tRNA charged with Sep. Therefore, the team engineered a second protein, EF-Tu, to directly deliver phosphoserine-charged tRNA to the ribosome, increasing the overall efficiency of the process. In this manner, the team successfully expanded the genetic code of the E. coli, allowing the incorporation of phosphoserine into any protein. Now, proteins may be studied in their activated states, such as cancer-related cells characterized by high levels of protein activation.

With this crucial information, this innovative Yale research team now intends to use the genetic code to engineer live proteins in states linked to cancer, hypertension, and type 2 diabetes. They stress, however, that this method of protein engineering has broader utility and can potentially create many more type of modified proteins.